Bioinformatics, expression analysis, and functional verification of allene oxide synthase gene HvnAOS1 and HvnAOS2 in qingke

Abstract Allene oxide synthase (AOS) is a key enzyme involved in the jasmonic acid (JA) synthesis pathway in plants. To explore its function on the regulatory mechanism of JA synthesis, we screened and identified two AOS genes HvnAOS1 and HvnAOS2 in qingke. Both HvnAOS1 and HvnAOS2 contained conserved heme-binding motif, which is most closely related to AtsAOS2, indicating controlled dehydration of fatty acid hydroperoxides to allene oxides. Molecular docking simulations identified the key amino acid sites that were important for heme binding and interaction with 13(S)-HPOT, respectively. The expression pattern also indicated that HvnAOS1 and HvnAOS2 were highly induced by JA, abscisic acid, and salicylic acid. Subcellular localization of HvnAOS1 and HvnAOS2 using transient expression of Agrobacterium tumefaciens showed the green fluorescent protein signal in the cell cytoplasm of the N. benthamiana leaves. Overexpression of HvnAOS1 and HvnAOS2 in Arabidopsis aos mutant restored male fertility and plant resistance to Botrytis cinerea, indicating that HvnAOS1 and HvnAOS2 can restore the functions of AOS in Arabidopsis aos mutant.


Introduction
Qingke (Hordeum vulgare L. var.nudum Hook.f.) is the most important grain and fodder crop, with the advantages of early maturity, easy cultivation, and tolerance to multiple adversities in the Qinghai-Tibetan Plateau of China [1][2][3].Qingke seeds are rich in nutrients and can be processed into a variety of foods, and are also an important raw material for winemaking, which is popular among people in the Qinghai-Tibetan Plateau [4][5][6].Due to the extremely harsh environment in the Qinghai-Tibetan Plateau region, such as drought, salinity, low temperature, and strong ultraviolet radiation, the production of qingke is subject to long-term comprehensive adversity stresses [4,7,8].So, how to improve the resistance to integrated adversities in the plateau region is of great significance for qingke production in the Qinghai-Tibetan Plateau.
As a kind of growth regulator, jasmonic acid (JA) and its derivatives (JAs) play an important role in plant growth and development and various physiological activities [9][10][11][12].In addition, JA can act as the most responsive signaling molecules to be in response to comprehensive stress, such as drought, salinity, high and low temperature, pests and diseases, mechanical damage, strong UV radiation, etc. [13][14][15].Allene oxide synthase (AOS) is the key enzymes in the JA synthesis pathway, and its substrate is 13(S)-hydroperoxy-linolenic acid (13(S)-HPOT).The core region of AOS contains a heme molecule, and after 13(S)-HPOT enters the core region of AOS, it is converted to unstable 12(S),13(S)epoxy-linolenic acid (12(S),13(S)-HPOT) catalyzed by heme and then converted by propylene oxide cyclase (POC) to 12-oxo-phytodienoic acid (12-OPDA) [16].12-OPDA eventually produces JA after a series of reactions, which is a key step in the synthesis of JA in plants.Many studies have shown that the AOS in plants is closely related to the regulation of plant resistance to drought, salinity, low temperature, pests and diseases, strong UV radiation, and other adversities [17][18][19][20].Many studies have shown that the expression of AOS genes are induced by various factors such as gibberellin (GA), abscisic acid (ABA), low temperature, salt, and drought stress, and they also play an important role in plant growth and development and in response to adversity stress [21][22][23][24].There are two AOS homologs, AOS1 and AOS2, in some plants, and the functions of AOS1 and AOS2 are the same, and they have different modes of action and different expression patterns [25].
In this study, we performed bioinformatics, expression analysis, and functional verification of AOS genes HvnAOS1 and HvnAOS2.This study provides new sights for the function of HvnAOS1 and HvnAOS2 on the regulation of JA synthesis under integrated stress resistance in qingke.

Expression pattern analysis of HvnAOS1 and HvnAOS2
To analyze the expression levels of HvnAOS1 and HvnAOS2 under different phytohormone treatments, the qingke seeds were planted in soil-based seedling boxes and treated with hormones when they reached the three-leaf stage.A 100 µM solution of ABA, methyl jasmonate (MeJA), GA, salicylic acid (SA), 6-benzyl aminopurine (6-BA), and naphthalene acetic acid (NAA) was prepared and 5 mL of each solution was sprayed with 1‰ Tween-20, while the control plants were sprayed with only 200 mL of pure water with 1‰ Tween-20.
The leaves were harvested and flash frozen in liquid nitrogen at 0 (control), 6, 12, 24, and 48 h after treatment and were stored at −80°C for RNA extraction.At least three biological replicates of each sample were performed to analyze the expression patterns of HvnAOS1 and HvnAOS2 [28,29].Total RNA from the leaves was extracted using the TransGen Transzol Up Plus kit.The cDNA was synthesized using the first-strand cDNA synthesis super mix kit (Transgen Biotech, Catalog No. AE301-02).The pair of specific primers of HvnAOS1 and HvnAOS2 were as follows: HvnAOS1: forward, TTCGTCGGCGACCGGTTC and reverse, CTGCCACACCGGACG; HvnAOS2: forward, ACCGGTTTGTCGGGG and reverse, CCCCA GGACCGGAT.The primers of reference gene 18SrRNA were as follows: forward, CGGCTACCACATCCAAGGAA and reverse, GCTGGAATTACCGCGGCT.The qRT-PCR reaction system consisted of 1.0 μL primers (10 μM), 2.0 μL cDNA, 10 μL Thunderbird SYBR qPCR Mix, and 6.0 μL ddH 2 O.The formula 2 −ΔΔCt was used for qRT-PCR analysis and each reaction was repeated three times.

Transformation of Arabidopsis and identification of transgenic plants
To efficiently test whether the HvnAOS1 and HvnAOS2 are functionally similar to Arabidopsis AOS, we transformed HvnAOS1 and HvnAOS2 into the Arabidopsis aos mutant to test whether HvnAOS1 and HvnAOS2 can complement the JA-synthase-related phenotypes of aos, respectively.Recombinant vectors pBI221:HvnAOS1-GFP and pBI221:HvnAOS2-GFP were transformed into Agrobacteria strain GV3101 and then infected inflorescence of Arabidopsis, respectively.Since Arabidopsis aos is male-sterile [30,31], we sprayed aos with 100 µM MeJA every 3 days to restore fertility for Agrobacterium (GV3101)mediated floral dip genetic transformation.Arabidopsis was cultured as described by An et al. [17].Harvested Arabidopsis seeds were sterilized with bleach for 5 min and then washed 5-6 times with sterilized water.These seeds were selected on Basta (20 mg L −1 ) medium for the positive transgenic seedlings.
The first generation of transgenic plants (T 1 ) was confirmed by PCR amplification using HvnAOS1 and HvnAOS2-specific primers, and T 2 or T 3 seeds were used for further research.

Identification of male fertility and Botrytis cinerea resistance in transgenic plant
Transgenic seedlings were grown on 1/2 MS medium containing 0.8% agar, 1% sucrose, and 20 mg L −1 Basta, wildtype (WT) seedlings were grown on same medium without Basta.After germination, the WT and transgenic Arabidopsis seedlings of similar size were selected and transferred to plant in 0.35-L pots with artificial mixed soil (pindstrup substrate: organic substrate: vermiculite = 5:4:1).The pots were placed under 12 h day and 12 h night cycles at 22°C for 25 days for the identification of male fertility and resistance.Arabidopsis and B. cinerea culture and spore suspension preparation were conducted as described by An et al. [17].Three leaves of similar size were taken from WT, aos, aos/HvnAOS1, and aos/HvnAOS2.The leaves were inoculated with 10 μL of 10 7 mL −1 spore suspension solution (0.025% Tween-20) and 15 μL control solution (autoclaved water and 0.025% Tween-20).The leaves were placed in a 90 mm diameter petri dish lined with moistened filter paper and sealed to keep the moisture in.Petri dishes were stored at 22°C.The diameter of the spots was counted after 2 days post-infection.

Results
3.1 Bioinformatics analysis of HvnAOS1 and HvnAOS2 genes

Cis-acting elements analysis of the promoter region
The amplified promoter region sequences of HvnAOS1 and HvnAOS2 gene were analyzed by the PlantCARE software.
A large number of TATA-box and CAAT-box promoter core  elements as well as light-responsive cis-acting elements were found in the promoter regions.Only three ABA, one growth hormone, and one gibberellin responsive cis-acting elements were found in HvnAOS1 promoter region (Table 1).
In addition to cis-regulatory elements of anaerobic induction, fenestra cell differentiation, seed-specific regulation, and circadian rhythm control were also found in the promoter of HvnAOS2 genes (Table 1).Subcellular localization prediction revealed that HvnAOS1 and HvnAOS2 were localized in the endoplasmic reticulum (Table 2).

Phylogenetic tree construction
The homologous protein sequences of HvnAOS1 and HvnAOS2 from qingke and 17 other plants were analyzed and their phylogenetic tree was constructed [32,33].The results of phylogenetic tree showed that HvnAOS1 and HvnAOS2 had the highest similarity to AtsAOS2 from Aegilops tauschii subsp.strangulata (Figure 1).

Amino acid sequence alignment and molecular docking simulation
The protein sequence alignment of HvnAOS1, HvnAOS2, and other plant AOS homologs were performed using DNAMAN 7.0.The results showed that all AOS proteins have the conserved heme binding key region (Figures 2  and 3, red region), the substrate catalysis key site, and heme Fe 3+ interactions key site (Figure 2).Protein binding cavity analyses revealed that the binding cavities of both  HvnAOS1 and HvnAOS2 were located within the protein's internal structure (Figure 4a and b [brown zone]).Molecular docking simulations revealed that one carboxyl group on heme in HvnAOS1 forms 2.0 Å hydrogen bonds with residues ASN420 and GLN353, respectively, and another carboxyl group on heme forms 2.2 Å hydrogen bonds with residue LYS432.The 13(S)-HPOT hydroxyl atom in HvnAOS1 forms 2.7 Å hydrogen bonds with residue ASN283 and 2.0 Å with residue and the Fe 3+ in heme in HvnAOS1 forms a 2.2 Å covalent bond with the sulfhydryl sulfur atom of residue CYS434.However, a carboxyl group on heme in HvnAOS2 forms a 2.3 Å hydrogen bond with residue ASN414, and another carboxyl group on heme forms a 2.0 Å hydrogen bond with residue VAL345.The 13(S)-HPOT hydrogen atom in HvnAOS2 (S)-HPOT hydroxyl hydrogen atom forms a 3.4 Å hydrogen bond with residue ASN278 and a 1.9 Å hydrogen bond with residue LYS284.The Fe 3+ in heme forms a 2.8 Å covalent bond with the sulfhydryl sulfur atom of residue CYS428.These interactions are essential for HvnAOS1 and HvnAOS2 to bind the substrate 13(S)-HPOT and the catalytic factor heme (Figures 3 and 5).

Subcellular localization of HvnAOS1 and HvnAOS2
To understand the localization of HvnAOS1 and HvnAOS2 in plant, HvnAOS1:GFP and HvnAOS2:GFP were transiently expressed in protoplast.Subcellular localization of HvnAOS1 and HvnAOS2 revealed that the GFP signal was observed in cytoplasm and fibrillary.In summary, these results suggested that HvnAOS1 and HvnAOS2 were accumulated in the endoplasmic reticulum in combination with the results of prediction (Figure 6).

Expression pattern analysis of HvnAOS1 and HvnAOS2 by plant hormone treatment
In view of many cis-acting elements related to plant hormones in HvnAOS1 and HvnAOS2 promoter region, we tested whether HvnAOS1 and HvnAOS2 were induced by plant hormones in the leaves.The results showed that HvnAOS1 and HvnAOS2 genes were strongly induced in the leaves with JA, ABA, and SA treatments, but there was no significant induction in the leaves with GA, NAA, and 6-BA treatments (Figure 7).

Over-expression of HvnAOS1 and HvnAOS2
in Arabidopsis aos mutant restores male fertility and causes immunity recovery against B. cinerea To efficiently test whether HvnAOS1 and HvnAOS2 are functional as AOS, we transformed HvnAOS1 and HvnAOS2 gene into the Arabidopsis aos mutant, respectively, and tested whether HvnAOS1 and HvnAOS2 can complement the JAinsensitivity-related phenotypes of aos.As described earlier, Arabidopsis aos mutant exhibited male sterility (Figure 8).However, over-expression of HvnAOS1 and HvnAOS2 in Arabidopsis aos mutant restored its male fertility, indicating that HvnAOS1 and HvnAOS2 are capable of the function on male fertility.JA is one of the major defense hormones in plants, and the Arabidopsis JA biosynthesis mutant aos are highly susceptible to the necrotrophic pathogen B. cinerea.In this study, we tested whether HvnAOS1 and HvnAOS2 are able to complement the function on defense against B. cinerea in the aos mutant.When inoculated B. cinerea on Arabidopsis leaves, the aos mutant exhibited high susceptibility to B. cinerea (Figure 9).However, the HvnAOS1 and HvnAOS2 complementation on the aos mutant were similar to the wild type Arabidopsis in terms of resistance (Figure 9).Taken together, HvnAOS1 and HvnAOS2 share the similar function with Arabidopsis AOS gene on male fertility and plant resistance to B. cinerea.

Discussion
AOS is one of the crucial synthase enzymes in the JA biosynthesis pathway in plants, belonging to the CYP74A family within the cytochrome P450 supergene family [34][35][36][37].Due to its involvement in the JA-related stress responses, growth developmental regulation, and hormone signaling pathways in plants, AOS genes have been a prominent subject of research in different plant species.Additionally, AOS plays distinct physiological roles in plants, further highlighting its significance.As a result, AOS remains a focal point of investigation, with numerous unknown functions awaiting discovery [38].There are currently very few studies on the structure, expression pattern, and function of AOS, and only a few studies have been reported in Arabidopsis and rice, so it is particularly important to study the structure, expression pattern, and function of HvnAOS1 and HvnAOS2.
In this study, we cloned HvnAOS1 and HvnAOS2 genes and their promoter regions, and analyzed their bioinformatics, expression patterns, and functions.The elements in the promoter regions of HvnAOS1 and HvnAOS2 are significantly different.They are likely to have different expression patterns and functions under different conditions.The HvnAOS1 promoter region contains a number of phytohormone-responsive cis-elements, whereas the promoter region of HvnAOS2 lacks phytohormone-responsive elements.Instead, it contains specific cis-regulatory elements related to anaerobic induction, mesophyll cell differentiation, seed-specific regulation, and diurnal rhythm control.HvnAOS1 is likely to be involved in JA synthesis under normal barley conditions or in common stress responses.On the other hand, HvnAOS2 may play a role in JA synthesis in specific tissues or under specific conditions.HvnAOS1 and HvnAOS2 have similar physicochemical properties, but HvnAOS1 is hydrophilic whereas HvnAOS2 is hydrophobic.
The predication results indicated that HvnAOS1 and HvnAOS2 were found to be located in the endoplasmic reticulum.However, some studies have found that the subcellular localization of AOS were different among different plants.Most plant AOS possess typical chloroplast transit peptides and are primarily located in chloroplasts.However, recent studies indicate that barley AOSs does not have chloroplast transit peptides, and AOSs in some plants can be detected within specific organelles such as PhAOS from Parthenium hysterophorus L. which is localized in intracellular rubber particles [39,40].
Molecular docking simulation of the key residues in the different interaction patterns of HvnAOS1 and HvnAOS2 indicated that HvnAOS1 interacts with 13(S)-HPOT at residue ASN283 and with heme Fe 3+ at residue CYS434.HvnAOS2 interacts with 13(S)-HPOT at residue ASN278 and with heme Fe 3+ at residue CYS428.Interestingly, these two key interaction sites are the same amino acid residues in both HvnAOS1 and HvnAOS2.Li et al. [16] investigated the crystal structure of PaAOS and iron porphyrin from Parthenium argentatum and its complex with the substrate analogue 13(S)-HODE and its intermolecular interactions, and found that the ASN-276 residue in PaAOS is in the active site and very close to heme, and that this amino acid site is highly conserved among AOS from different species, ASN-276 is thought to play an important role in substrate catalysis, and the CYS-426 residue forms a covalent bond with Fe 3+ in the center of heme, which is thought to play an important role in the binding to heme.The results in this study are consistent with the reports, which suggest that these interactions are highly conserved in AOS and are important sites of action for AOS function.In addition, HvnAOS1 and HvnAOS2 are capable of complementing the Arabidopsis aos mutant, suggesting that HvnAOS1 and HvnAOS2 have similar functions as the Arabidopsis AOS gene.
In conclusion, we have identified two AOS genes HvnAOS1 and HvnAOS2 from hulless barley "Kunlun 15" that controlled the dehydration of fatty acid hydroperoxides to allene oxides.HvnAOS1 and HvnAOS2 were highly induced by JA, ABA, and SA.HvnAOS1 and HvnAOS2 mainly accumulated in the cell cytoplasm of the N. benthamiana leaves.Overexpression of HvnAOS1 and HvnAOS2 in Arabidopsis aos mutant restored male fertility and plant resistance to B. cinerea, indicating that HvnAOS1 and HvnAOS2 can restore the functions of AOS in Arabidopsis aos mutant.This study will provide a reference for research on the acid oxydase synthase and regulation of JA synthesis in qingke.It is important to note that the specific AOS genes and their functions can vary among different plant species, and further research is needed to explore the diversity and functional significance of AOS genes in various plants.

23 TC
element conserved DNA module involved in light responsiveness 2 CAAT-box CAAAT; CCAAT Common cis-acting element in promoter and enhancer regulatory element involved in zein metabolism regulation 2 TATA-box TATA; TATAA; ATATAA; TATATA; TATATAAATC Common cis-acting element in promoter and enhancer regions 29 regulatory element involved in circadian control 1

Figure 2 :
Figure 2: Protein sequence alignment of HvnAOS1 and HvnAOS2.*: The heme Fe 3+ interactions key site for HvnAOS1 and HvnAOS2; •: The key amino acid site for substrate catalytic.

Figure 5 :
Figure 5: Key amino acid sites analysis of the HvnAOS1 and HvnAOS2 interact with heme and 13(S)-HPOT.(a) Key amino acid sites analysis of the HvnAOS1 interact with heme and 13(S)-HPOT.(b) Key amino acid sites analysis of the HvnAOS2 interact with heme and 13(S)-HPOT.

Table 1 :
Analysis of cis-acting elements in

HvnAOS2 Hordeum vulgare L. var. nudum Hook. f. XP_044982338.1
Figure 1: Phylogenetic tree of HvnAOS1 and HvnAOS2 with other species.Phylogenetic analysis of HvnAOS1 and HvnAOS2 with its homologs from other species.Phylogenetic analysis of AOS from dicotyledon and monocotyledon plants using MEGA7.Branches are labeled with GenBank accession numbers and the organisms.points of 8.93 and 7.65, and instability indices of 33.75 and 32.20, respectively.The α-helix, extended strand, β-turn, and random coil of HvnAOS1 and HvnAOS2 proteins accounted for 39.43, 15.61, 5.43, 39.36, and 40.62% of the total amino acids, respectively, and 39.36, 40.62, 15.62, 4.38, 39.38%, respectively.